Small survival-promoting/immunomodulatory peptide for treatment of brain damage, neurodegenerative disorders, and inflammatory disorders

ABSTRACT

A synthetic peptide sequence demonstrating neuroprotective and anti-inflammatory functions is disclosed. Methods of use for the synthetic peptide are also provided.

[0001] This application claims priority to U.S. Provisional Application60/426,536, filed Nov. 15, 2002, the entire contents of which areincorporated herein by reference.

[0002] Pursuant to 35 U.S.C. §202(c), is acknowledged that the U.S.Government has certain rights in the invention described, which was madein part with funds from NIH grant number NS16347, from the NationalInstitute of Neurological Disorders and Stroke.

FIELD OF THE INVENTION

[0003] The present invention relates to a composition of mattercomprising a small peptide, for promoting neurite outgrowth, enhancingsurvival of neuronal cells, and/or inhibiting phospholipase A2; to apharmaceutical preparation containing the small peptide; and to its usein the treatment of neuron damage, neurodegenerative disorders, andneuronal and non-neuronal disorders with an inflammatory component.

BACKGROUND OF THE INVENTION

[0004] Several publications and patent documents are cited in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

[0005] Neurotrophic factors are considered to be vital for normaldevelopment of the nervous system. During development, neuronal targetstructures produce limited amounts of specific neurotrophic factorsnecessary for both the survival and differentiation of neuronsprojecting into the structures. The same factors have been found to beinvolved in the survival and/or maintenance of mature neurons.

[0006] A neurotrophic factor is defined as a substance capable ofincreasing and/or maintaining survival of a neuron population, andpossibly affecting outgrowth of neurites (neuron processes) and certainother metabolic activities of a neuron. Neurotrophic factors aregenerally described as soluble molecules synthesized in the peripheraltargets of neurons and transported to their cell bodies, where theyexert their effects.

[0007] Studies with isolated neurotrophic factors have shown thatexogenously added neurotrophic factors can exert their neurotrophiceffects upon cultured neurons in vitro, or by administration to damagedor degenerated neurons in vivo. For this reason, various neurotrophicfactors have received great attention as potential therapeutic agentsfor treatment of degenerative diseases of the central nervous system, aswell as traumatic damage to the CNS. For example, nerve growth factor(NGF) has been shown to increase the survival, function and regenerationof cholinergic neurons in the basal forebrain. Degeneration of thispopulation of cholinergic neurons has been associated with patientshaving Alzheimer's disease, and could be the primary neuronal defectresponsible for the loss of cognitive function associated withAlzheimer's disease. NGF has been found to be synthesized and releasedfrom the target areas of these cholinergic neurons in the hippocampusand neurocortex, both areas of the brain associated with learning andmemory. See Springer, J. E., Drug News and Perspectives, 4: 394-99(1991). As another example, a dopaminergic neurotrophic factor (DNTF)has been purified and characterized, and found to promote survival andneurite outgrowth of dopaminergic neurons of the substantia nigra. DNTFis considered a potentially valuable therapeutic agent for the treatmentof Parkinson's disease which involves degeneration of dopaminergic motorneurons of the central nervous system (U.S. Pat. No. 5,215,969 toSpringer et al., 1993).

[0008] It can be seen from the foregoing examples that neurotrophicfactors are a valuable source of therapeutic agents for the treatment ofneuron damage and neurodegenerative disease. However, the development ofsuch factors as therapeutic agents can be problematic. For example, itis difficult to determine the specificity of an endogenous neurotrophicagent, i.e., whether different factors exist for different nervoussystem pathways, and which neuron populations in those pathways areaffected by a factor. In fact, many identified neurotrophic agents havebeen shown to have a wide range of biological functions, acting on bothcentral and peripheral neurons, as well as non-neuronal cells in vitro(e.g., polypeptide growth factors and ciliary neurotrophic factor,CNTF). In the central nervous system, with its complex interconnectionsand heterogeneous neuron types, it is difficult to determine whichneurotrophic factors are effective on a particular neuronal population.This difficulty is further exacerbated by the fact that many of theneurotrophic factors that have been characterized have been found to beclosely related to one another. For example, it is now known that NGFpossesses amino acid sequence homology to brain-derived neurotrophicfactor (BNDF), a protein with similar, but not identical, in vitroproperties as NGF (Barde et al., EMBO J., 1: 549-53, 1982; Leibrock etal., Nature, 341: 149-52, 1989). In fact, NGF, BNDF and the neurotrophin(NT) series have been classified as members of a superfamily ofneurotrophic factors (NGF superfamily). Because of their similarity inamino acid sequence (and hence nucleotide sequences encoding thefactor), it has been difficult to develop nucleic acid or antibodyprobes that are specific for a particular member of the family. The lackof a specific means for identifying a particular neurotrophic factor hashindered the elucidation of particular neuronal populations affected bya specific factor.

[0009] An additional obstacle to developing neurotrophic factors astherapeutic agents for treatment of damaged neurons is that few in vivomodels exist to study the survival-promoting activity of these factorsin the central nervous system. In order to develop a neurotrophic factoras an effective therapeutic agent for the treatment of neurondegeneration, it is important to be able to determine where in thecentral nervous system the neurotrophic factor operates, whether thetreatment with exogenous neurotrophic factor is effective, and theconcentration of neurotrophic factor effective for imparting atherapeutic effect. Such an objective would best be accomplished with aneurotrophic factor that is identifiable and distinct from otherfactors, that is capable of exerting an effect on many different neuronpopulations, and for which in vivo models are available to test theefficacy of the neurotrophic factor on a specific neuron population.

[0010] The neuron survival-promoting peptide Y-P30 was originallyidentified in the secretions of neural cells (neuroblastoma andretinoblastoma) subjected to oxidative stress (Cunningham, et al. 1998).Partially purified fractions of conditioned culture medium were screenedin vitro until the active Y-P30 peptide was identified—the syntheticversion of this peptide was then tested in vitro and in vivo and foundto support neural cells which were degenerating for a variety ofreasons, including oxidative stress and central nervous system trauma(Cunningham, et al. 1998; 2000). This peptide was later confirmed to bepart of an endogenous human polypeptide (˜12 kiloDaltons) named DSEPafter identification of the human cDNA encoding DSEP and the locus ofthe DSEP gene in human chromosomal region 12 q (Cunningham, et al.2002). In that study, it was found that overexpression of the fulllength polypeptide in neural cells made them resistant to several formsof oxidative stress including that resulting from immune cell attack.

[0011] The contribution of inflammatory cells and their secretions tocell death after CNS injury or in neurodegenerative disorders is for themost part well established (Stoll, 1998). The principal immune cellparticipants in the response to traumatic CNS injury are monocytederivatives (microglia/macrophages). These cells are the source of anumber of inflammatory agents that may contribute to neuron death,including superoxide anion, nitric oxide, IL-1β, and TNFα (reviewed byRothwell, et al 1996, Stoll et al 1998, Jander, et al 1998, 2000; andTurrin, et al 2001). TNFα is best known for its cytotoxic activityoutside the nervous system, but also has pronounced toxic activity onneural cells after brain injury (Barone, et al, 1997; Lavine, et al1998). Both overexpression of the full length DSEP molecule andapplication of Y-P30 inhibits the appearance and differentiation ofmacrophages and microglia (Cunningham et al. 1998, 2000, 2002).

[0012] Steriod anti-inflammatory drugs currently used to treat nervoussystem injury and other disorders with an inflammatory component operatein part by stimulating the production of endogenous inhibitors ofphospholipases (A2) (PLA2) which are the enzymes responsible for theproduction of several lipid mediators of inflammation (Flower, R J etal. 1979). PLA2 enzymes and downstream participants in this pathway playa role in chronic neurodegenerative disorders including Alzheimer'sdisease (Farooqui A A, et al., 1999; Hull M, et al., 2002).

[0013] Therefore, a need exists in the art for identification andtesting in vivo of new neurotrophic factors which are distinct fromother factors, exert an effect on many different neurons, and/or whichcan act as PLA2 inhibitors, to facilitate the development of newtherapies for neurodegenerative disorders and for other diseases with aninflammatory component.

SUMMARY OF THE INVENTION

[0014] In accordance with one aspect of the present invention, there isprovided a synthetic peptide, CHEC-9, having the sequence of CHEASAAQC(SEQ ID NO: 1), or variants thereof, wherein the peptide promotes neuronsurvival, inhibits a brain's immune response to degenerating elements,and/or inhibits phospholipase A2. The peptide may be linear or cyclized.

[0015] In accordance with another aspect of the present invention, thereis provided a pharmaceutical preparation comprising the syntheticpeptide, CHEC-9.

[0016] In another aspect of the invention, there is provided a nucleicacid encoding a synthetic peptide, CHEC-9, having the sequence ofCHEASAAQC (SEQ ID NO: 1), or variants thereof.

[0017] In yet another embodiment of the invention, there is an antibodyor fragment thereof which is immunologically specific for a syntheticpeptide, CHEC-9, having the sequence of CHEASAAQC (SEQ ID NO: 1), orvariants thereof.

[0018] In accordance with yet another aspect of the present invention,there is provided a method for treating a patient having aneurodegenerative disorder by administering to the patient atherapeutically effective amount of CHEC-9. Such neurodegenerativedisorders include, but are not limited to, (1) trauma, (2) stroke, (3)nonspecific anoxia (i.e., anoxia due to drowning, suffocation, etc.),(4) neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease and amyotrophic lateral sclerosis (ALS); and (5) mentalretardation syndromes associated with progressive neuronal degeneration(e.g., cerebral palsies).

[0019] In accordance with another aspect of the present invention, thereis provided a method for treating a patient having a disorder with aninflammatory component, by administering to the patient atherapeutically effective amount of CHEC-9. Such disorders include, butare not limited to, (1) asthma; (2) autoimmune disorders; (3) allergies;(4) arthritis; and (5) any disorder which might benefit from treatmentusing a steroid or a phospholipase A2 inhibitor.

[0020] In accordance with yet another aspect of the invention, a kit isprovided which facilitates administering or testing for the CHEC-9peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows increased survival of SY5Y neuroblastoma cellsexposed to CHEC-9, following medium change and serum deprivation for 48hrs. The cells were seeded at low density in serum and changed to serumfree medium (with added CHEC-9 peptide or vehicle) after 2 hrs. Cellsurvival was measured with the WST electrocoupling reagent (Ocinda). Thegraph on the left shows increased CHEC-9 concentration correlates withincreased cell survival. Three representative cultures from the 2 groupsare shown in the panels on the right. Coomassie Blue stain. Bar=100 μm.(p value based on n=16 cultures for each condition in 2 separateexperiments.)

[0022]FIG. 2 shows the coronal section through the cerebral cortex ofrats that received stab wounds in area 3. The rats survived for 4 daysfollowing the lesion after which their brains were processed for cresylviolet staining or immunostaining with macrophage/microglia marker ED-1in adjacent sections (inset). The vehicle treated animal shows a typicalresponse to the lesion including a pronounced invasion of inflammatorycells and degeneration of cortical tissue. Systemic treatment withCHEC-9 inhibits both processes. Bar=1 mm.

[0023]FIG. 3 shows microglia cells which were purified from neonatalrats, activated with 100 nM retinoic acid (RA) on days 1 and 2 in vitro,and examined on day 3 or 4. It is shown that TNFα immunoactivity isreduced in these cells that were treated with 1 nM CHEC-9 during theperiod of RA activation.

[0024]FIG. 4 is a graph showing that percent concentrations ofmicroglia/macrophages at the dorsal and ventral margins of the lesions 4days after stab wounds to the parietal cortex of rats, are lower inanimals which were administered the CHEC-9 peptide. Near marginal orwhite matter layers, where ameboid cells appeared most consistently inboth groups, the number was reduced by 75% (N=6 in each group,p=4×10−3). Such cells are very sparse in the midportions of the lesionsafter peptide treatment.

[0025] FIGS. 5A-D show that CHEC-9 treatment inhibits PLA2 enzyme andrelated activities. (A). CHEC-9 inhibition of phospholipase A2 from beevenom is maximal within the first 2 min of the reaction at a variety ofpeptide concentrations. (B). Representative Michaelis-Menton Plots plotusing 5% serum samples from CHEC-9 and control-injected rats. The bottomplot is from a pair of rats in which one was injected with CHEC-9 andone with a peptide where the positions of glutamate and adjacent alaninewere switched (CHAC-9). The dissociation constants are noted on theplots. (C). CHEC-9 treatment inhibits platelet aggregation. Plateletswere isolated from untreated rats and incubated with 0.1 nM CHEC-9 orequivalent tris solvent in HBSS (left graph), or from rats treated with100 μg CHEC-9 or vehicle (right graph). Rates of aggregation weredetermined over a period 5-30 minutes after addition of the indicatedconcentrations of PMA (**p<0.01, ***p<0.001. n=10 each in 2 directtreatment and 2 injection experiments). Micrographs of CHEC-9 andcontrol treated platelets at the end of one of the experiments areshown. Bar=100 μm. (D). CHEC-9 treatment results in a typical migrationof platelet sPLA2 IIa on SDS gels. Isolation and washing of plateletsprior to above analysis caused release of sPLA2 IIa which migrates, asexpected, with an apparent molecular weight of ˜14 kD on SDS gels rununder reducing conditions (lanes 1,3 control, treated). SPLA2 releasedfrom platelets treated with CHEC-9 either 100 μg injected into theanimal, (lane 2), or added directly (0.1 nM) to platelets isolated fromuntreated rats (lane 4) shows strong sPLA2 IIa bands that run withhigher apparent molecular weights. This suggests that treatment hadmodified the sPLA2 IIa enzyme structure and/or promoted the formation ofstabilized enzyme complexes or aggregates.

[0026] FIGS. 6A-C show that anti-YP30 antibody produces increasedcortical lesion size and sera toxicity. (A) Coronal sections throughmedial part of cerebral cortex seven days after a lesion in area 2 ofrats immunized against DSEP-KLH or KLH carrier protein. The lesions aremade by placing a 1 mm³ piece of gelfoam on the cortical surface. Thereare accumulations of cells at the margins of the lesions (large arrowand arrowheads) many of which are microglia/macrophages. At the 7 daysurvival, the lesions are considerably larger in rats immunized againstDSEP, as can be seen in the photomicrographs and graph of volumemeasurements from serial sections (p<0.001 at 7 days, n=9; n.s.d at 4days, n=6; A, B). Note also that the lesion in the anti-DSEP rat appearsto have expanded from the original boundaries (arrowheads) into theadjacent parenchyma and white matter leaving behind a large cyst. Bar=1mm. (C) Graph showing killing of SY5Y and HN33.1 cells by DSEP antisera.The cells were treated with 5% serum from rats immunized againstDSEP-KLH conjugate or KLH alone (protein concentrations wereequivalent). Viability was measured with the WST electrocoupling reagent72 hrs after treatment and expressed as a percentage of the controlvalue (anti-KLH). Cells treated with DSEP antisera degenerate whilethose treated with anti-KLH do not.

DETAILED DESCRIPTION OF THE INVENTION

[0027] A nine amino acid peptide CHEASAAQC (SEQ ID NO: 1, designatedCHEC-9 or CH-QC9) has been identified, synthesized and used to promotesurvival of neural cells in vitro and in vivo, including after cerebralcortex injury. The peptide rescues neurons that would usually shrink,die or disintegrate following traumatic brain damage. Furthermore, thepeptide posses demonstrable phospholipase A2 inhibitory activity, andtherefore has utility as a modulator of inflammation. A CHEC-9 peptidevariant having the sequence CAHAQAESC also promotes survival of neuralcells.

[0028] CHEC-9 constitutes an internal sequence of survival promotingpeptide Y-P30 (U.S. Pat. No. 6,262,024). CHEC-9 and Y-P30 are derivedfrom a 12 kiloDalton endogenous human protein, DSEP (GenBank Accession#AY044239, T. J. Cunningham, et al., 2002). CHEC-9 and Y-P30 differ fromthe sequence of DSEP in having a cysteine at position 23, instead oflysine. Like the larger peptides, CHEC-9 to promotes neuron survival andinhibits aspects of the immune response to cerebral cortex lesions, inparticular the appearance and invasion of macrophages and microglia atthe site of injury. Accordingly the peptide may be used for treatment ofdisorders involving acute neural degeneration (stroke and traumaticbrain damage), as well as for treatment of several chronicneurodegenerative disorders including Alzheimer's disease. In the latterapplications, CHEC-9 inhibits both neuron death and the brain's immuneresponse to degenerating elements, which should slow the progress ofthese disorders and attendant decline of behavioral performance.Additionally, CHEC-9 inhibits phospholipase A2, and thus may be used totreat disorders associated with inflammation.

[0029] A “CHEC-9 peptide” is a peptide having the sequence of CHEASAAQC(SEQ ID NO: 1). The peptide may be linear or cyclic. The term “CHEC-9peptide” may include variants of SEQ ID NO:1, wherein as few as 1 or asmany as 9 amino acids are changed, provided that the peptide stillpromotes neuron survival, inhibits a brain's immune response todegenerating elements, and/or inhibits phospholipase A2. Variants mayhave mutations comprising insertions, deletions, or substitutions ofamino acids. Variants preferably comprise conservative amino acidsubstitutions.

[0030] A “conservative amino acid substitution” as defined herein refersto replacement of an amino acid with a functionally and biochemicallyequivalent amino acid. These substitutions provide similar or enhancedfunction of a peptide. Functionally-equivalent amino acids are aminoacids which share a common structure, side chain, polarity, and soforth. Examples of amino acids which may be functionally equivalent are:hydrophobic Ala, His, Ile, Leu, Met, Phe, Trp, Tyr, Val neutralhydrophilic Cys, Ser, Thr polar Asn, Gln, Ser, Thr acidic/negativelycharged Asp, Glu charged Arg, Asp, Glu, His, Lys basic/positivelycharged Arg, His, Lys basic Arg, Asn, Gln, His, Lys residues thatinfluence Gly, Pro chain orientation aromatic His, Phe, Trp, Tyr

[0031] An autoimmune disease is a disease which occurs when one or morecomponents of the immune system targets the cells, tissues, and/ororgans of a person's own body. Autoimmune diseases include, but are notlimited to Multiple sclerosis, Myasthenia gravis, Autoimmuneneuropathies such as Guillain-Barré, Autoimmune uveitis, InflammatoryBowel Disease (including Crohn's Disease and Ulcerative colitis) Primarybiliary cirrhosis, Autoimmune hepatitis, Type 1 or immune-mediateddiabetes mellitus, Autoimmune thyroid disease (including Grave's Diseaseand Hashimoto's thyroiditis), Autoimmune oophoritis and orchitis,Autoimmune disease of the adrenal gland, Autoimmune hemolytic anemia,Pernicious anemia, Autoimmune thrombocytopenia, Temporal arteritis,Anti-phospholipid syndrome, Vasculitides such as Wegener'sgranulomatosis, Behcet's disease, Rheumatoid arthritis, Systemic lupuserythematosus, Scleroderma Polymyositis, dermatomyositis,Spondyloarthropathies such as ankylosing spondylitis, Sjogren'ssyndrome, Psoriasis, Dermatitis herpetiformis, Pemphigus vulgaris, andVitiligo.

[0032] I. Preparation of Human CHEC-9-Encoding Nucleic Acid Molecules,CHEC-9 Peptides, and Antibodies Thereto

[0033] Nucleic Acid Molecules: Nucleic acid molecules encoding CHEC-9peptides of the invention may be prepared by two general methods: (1)synthesis from appropriate nucleotide triphosphates, or (2) isolationfrom biological sources. Both methods utilize protocols well known inthe art. Preparation of an isolated nucleic acid molecule of theinvention may be by oligonucleotide synthesis. The nucleic acidsynthesized may be any combination of codons which encode the CHEC-9peptide. Synthetic oligonucleotides may be prepared by thephosphoramidite method employed in the Applied Biosystems 38A DNASynthesizer or similar devices. The resultant construct may be purifiedaccording to methods known in the art, such as high performance liquidchromatography (HPLC). Alternatively, nucleic acid sequences encodingthe CHEC-9 peptide may be isolated from appropriate biological sourcesusing methods known in the art. Suitable probes for this purpose arederived from sequences which encode the amino acids of CHEC-9.

[0034] In accordance with the present invention, nucleic acids havingthe appropriate level of sequence homology with the CHEC-9 peptide maybe identified by using hybridization and washing conditions ofappropriate stringency. For example, hybridizations may be performed,according to the method of Sambrook et al., Molecular Cloning, ColdSpring Harbor Laboratory (1989), using a hybridization solutioncomprising: 5×SSC, 5× Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50%formamide. Hybridization is carried out at 37-42° C. for at least sixhours. Following hybridization, filters are washed as follows: (1) 5minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at roomtemperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in1×SSC and 1% SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS, changingthe solution every 30 minutes.

[0035] One common formula for calculating the stringency conditionsrequired to achieve hybridization between nucleic acid molecules of aspecified sequence homology (Sambrook et al., 1989) is as follows:

T_(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex

[0036] As an illustration of the above formula, using [Na⁺]=[0.368] and50% formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5°C. with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

[0037] The stringency of the hybridization and wash depend primarily onthe salt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated T_(m) of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the T_(m) of the hybrid. In regards tothe nucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5× Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon spermDNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15minutes.

[0038] Nucleic acids of the present invention may be maintained as DNAin any convenient cloning vector. In a preferred embodiment, clones aremaintained in a plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

[0039] CHEC-9-encoding nucleic acid molecules of the invention includecDNA, genomic DNA, RNA, and fragments thereof which may be single- ordouble-stranded. Thus, this invention provides oligonucleotides havingsequences capable of hybridizing with at least one sequence of a nucleicacid molecule of the present invention. As mentioned previously, sucholigonucleotides are useful as probes for detecting or isolating CHEC-9related nucleic acids.

[0040] It will be appreciated by persons skilled in the art thatvariants (e.g., allelic variants) of CHEC-9 sequences exist in the humanpopulation, and must be taken into account when designing and/orutilizing oligonucleotides of the invention. Accordingly, it is withinthe scope of the present invention to encompass such variants, withrespect to the CHEC-9 sequences disclosed herein or the oligonucleotidestargeted to specific locations on the respective genes or RNAtranscripts. Accordingly, the term “natural allelic variants” is usedherein to refer to various specific nucleotide sequences of theinvention and variants thereof that would occur in a human population.The usage of different wobble codons and genetic polymorphisms whichgive rise to conservative or neutral amino acid substitutions in theencoded protein are examples of such variants.

[0041] Additionally, the term “substantially complementary” refers tooligonucleotide sequences that may not be perfectly matched to a targetsequence, but such mismatches do not materially affect the ability ofthe oligonucleotide to hybridize with its target sequence under theconditions described.

[0042] Proteins: CHEC-9 peptide, and functional variants thereof may beprepared in a variety of ways, according to known methods. The peptidemay be synthesized using an automated peptide synthesizer.Alternatively, the peptide may be purified from appropriate sources,e.g., transformed bacterial or animal cultured cells or tissues, byimmunoaffinity purification. The availability of nucleic acid moleculesencoding CHEC-9 peptide enables production of the peptide using in vitroexpression methods known in the art. For example, a CHEC-9 encodingpolynucleotide may be cloned into an appropriate in vitro transcriptionvector, such as pSP64 or pSP65 for in vitro transcription, followed bycell-free translation in a suitable cell-free translation system, suchas wheat germ or rabbit reticulocyte lysates. In vitro transcription andtranslation systems are commercially available, e.g., from PromegaBiotech, Madison, Wis. or Gibco-BRL, Gaithersburg, Md.

[0043] Alternatively, larger quantities of CHEC-9 peptides may beproduced by expression in a suitable prokaryotic or eukaryotic system.For example, part or all of a DNA molecule, such as a nucleic acidencoding CHEC-9 may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli. Such vectors comprisethe regulatory elements necessary for expression of the DNA in the hostcell positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences.

[0044] The CHEC-9 peptide produced by gene expression in a recombinantprokaryotic or eukaryotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinantpeptide/protein is expressed and thereafter secreted from the host cell,and readily purified from the surrounding medium. Ifexpression/secretion vectors are not used, an alternative approachinvolves purifying the recombinant protein by affinity separation, suchas by immunological interaction with antibodies that bind specificallyto the recombinant protein or nickel columns for isolation ofrecombinant proteins tagged with 6-8 histidine residues at theirN-terminus or C-terminus. Alternative tags may comprise the FLAG epitopeor the hemagglutinin epitope. Such methods are commonly used by skilledpractitioners.

[0045] The human CHEC-9 peptide and functional homologs or variantsthereof, prepared by the aforementioned methods, may be analyzedaccording to standard procedures. For example, such proteins may besubjected to amino acid sequence analysis, according to known methods.One such peptide variant which also has neuron protective activity isthe peptide having the sequence CAHAQAESC.

[0046] The CHEC-9 peptide may be oxidized (cyclized), or akylated(lineraized).

[0047] Antibodies: The present invention also provides antibodiescapable of immunospecifically binding to peptides of the invention.Polyclonal antibodies directed toward CHEC-9 peptide may be preparedaccording to standard methods. In a preferred embodiment, monoclonalantibodies are prepared, which react immunospecifically with CHEC-9peptide.

[0048] Polyclonal and/or monoclonal antibodies may be prepared asdescribed in several laboratory protocol handbooks, and scholarlyjournals including: Köhler and Milstein, Nature, 256: 495-7 (1975);Molecular Cloning: A Laboratory Manual, Sambrook et al. eds., ColdSpring Harbor Laboratory Press (1989); Ausubel et al. (supra), andAntibodies: A Laboratory Manual, Harlow and Lane eds., Cold SpringHarbor Laboratory Press (1988).

[0049] Polyclonal or monoclonal antibodies that immunospecificallyinteract with CHEC-9 peptide may be utilized for identifying andpurifying CHEC-9 peptide. For example, antibodies may be utilized foraffinity separation of peptides with which they immunospecificallyinteract. Antibodies may also be used to immunoprecipitate peptides froma sample containing a mixture of peptides/proteins and other biologicalmolecules. Other uses of anti-CHEC-9 peptide antibodies are describedbelow.

[0050] Antibodies according to the present invention may be modified ina number of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus, the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

[0051] Exemplary antibody fragments, capable of binding an antigen orother binding partner, are Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)₂ fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

[0052] II. Uses of CHEC-9-Encoding Nucleic Acids, CHEC-9 Proteins andAntibodies Thereto

[0053] CHEC-9-Encoding Nucleic Acids: CHEC-9-encoding nucleic acids maybe used for a variety of purposes in accordance with the presentinvention. CHEC-9-encoding DNA, RNA, or fragments thereof may be used asprobes to detect the presence of and/or expression of nucleic acidsencoding CHEC-9 peptides. Methods in which CHEC-9-encoding nucleic acidsmay be utilized as probes for such assays include, but are not limitedto: (1) in situ hybridization; (2) Southern hybridization (3) northernhybridization; and (4) assorted amplification reactions such aspolymerase chain reactions (PCR). Thus, CHEC-9-encoding nucleic acids ofthe present invention may be used for detecting CHEC-9 in vitro or invivo.

[0054] Additionally, the nucleic acids of the invention may be used toidentify genes encoding proteins that interact with CHEC-9 peptides(e.g., by the “interaction trap” technique).

[0055] The CHEC-9 nucleic acids of the invention may be introduced intohost cells. In a preferred embodiment, mammalian cell lines are providedwhich comprise a CHEC-9-encoding nucleic acid or a variant thereof. Hostcells contemplated for use include, but are not limited to NIH3T3, CHO,HELA, yeast, bacteria, insect and plant cells. The CHEC-9 encodingnucleic acids may be operably linked to appropriate regulatoryexpression elements suitable for the particular host cell to beutilized. Methods for introducing nucleic acids into host cells are wellknown in the art. Such methods include, but are not limited to,transfection, transformation, calcium phosphate precipitation,electroporation and lipofection.

[0056] The host cells described above may be used as screening tools toidentify compounds that modulate CHEC-9 expression and/or activity.Modulation of CHEC-9 expression and/or activity may be assessed bymeasuring alterations in CHEC-9 mRNA or peptide levels in the presenceof the test compound.

[0057] As described above, CHEC-9-encoding nucleic acids are also usedto advantage to produce large quantities of substantially pure CHEC-9peptides, or selected portions thereof.

[0058] CHEC-9 Peptide: It has been discovered that CHEC-9 promotessurvival of neural cells in vitro and in vivo, and inhibitsphospholipase A2. Thus, peptide CHEC-9 and pharmaceutical preparationscomprising the same have broad utility in the treatment of neurondamage, neurodegenerative disease, and disorders with an inflammatorycomponent. The uses of these materials described hereinbelow areintended to exemplify their utility, and are not intended to limit theinvention.

[0059] Such neurodegenerative diseases and disorders include, but arenot limited to (1) trauma, (2) stroke, (3) nonspecific anoxia (i.e.,anoxia due to drowning, suffocation, etc.), (4) neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease andamyotrophic lateral sclerosis (ALS); and (5) mental retardationsyndromes associated with progressive neuronal degeneration (e.g.,cerebral palsies).

[0060] Disorders with an inflammatory component include, but are notlimited to, (1) asthma; (2) autoimmune disorders; (3) allergies; (4)arthritis; and (5) any disorder which might benefit from treatment usinga steroid or a phospholipase A2 inhibitor.

[0061] A pharmaceutical preparation of CHEC-9 is formulated foradministration to patients by combining the peptide with a biologicallyacceptable medium, such as water, buffered saline, orosmotically-adjusted media such as polyol (e.g., glycerol, propyleneglycol, liquid polyethylene glycol and the like) or suitable mixturesthereof. The term “biologically acceptable medium” includes allsolvents, dispersion media and similar components which may beappropriate for the selected route of administration of thepharmaceutical preparation. The use of such biologically acceptablemedia for pharmaceutical preparations is well known in the art. Unless aconventional medium or agent is incompatible with the active ingredientof CHEC-9, its use in the pharmaceutical preparation of the invention iscontemplated.

[0062] The pharmaceutical preparation is preferably administeredparenterally, by introduction into the central nervous system of thepatient. This may be accomplished by intracerebroventricular infusiontargeted to the location of neuron damage. Other methods, such assystemic administration via an i.v. may also be utilized to administer apharmaceutical preparation containing CHEC-9. Administration may be byany method that allows CHEC-9 to cross the blood/brain barrier, eitheralone or linked to a carrier, including injection into the bloodstream,subcutaneous or intramuscular injection, as well as oral, intranasal,rectal and ophthalmic administration. In a preferred embodiment,solutions comprising CHEC-9 may be injected subcutaneously.

[0063] CHEC-9 peptide may be administered topically or transdermally,such as in a cream, salve, spray, ointment, or dermal patch.

[0064] Alternatively, CHEC-9 peptides are incorporated into a solidmatrix, which can be implanted into regions of the nervous system/brainrequiring treatment. For example, a pre-determined concentration ofCHEC-9 may be mixed in equal parts with a 2% sodium alginate medium, andentrapped in the resulting gel matrix. The sodium alginate gel ispolymerized in the form of small beads by dropping the gel into a 0.5 MCaCl2 solution. Other solid or semi-solid biologically compatiblematrices are also contemplated for use in the present invention. Theseinclude various natural bio-polymers, such as xanthan and carob gums(See Mugnier et al., Appl. Environ. Microbiol., 50: 108-14 (1985).

[0065] The pharmaceutical preparation comprising CHEC-9 isadvantageously formulated in dosage units, which is defined herein as adiscrete unit of the pharmaceutical preparation appropriate for thepatient undergoing treatment. As used herein, the term “patient” refersto humans and animals. A dosage will contain the quantity of activeingredient determined to produce the desired therapeutic effect inconjunction with the selected pharmaceutical carrier.

[0066] The appropriate dosage of a pharmaceutical preparation comprisingCHEC-9 as the active ingredient may be determined by in vitro and invivo procedures. The optimum effective concentration of CHEC-9 isdependent upon the type of neuron being treated and the protocol andsource used for purification. Therefore, once the target neuronpopulation has been identified, the optimum effective concentration ofCHEC-9 may be determined by an in vitro assay. For example, a selectedneuron population may be grown in culture for 2-4 days in definedserum-free medium. Pre-determined concentrations of CHEC-9 in anappropriate biological medium is then added to the culture dishes every24 hours. After the incubation period, neurons and dendrites may beidentified by immunocytochemically, e.g., with an antibody against aneuron-specific marker, such as MAP2. Neuron survival and neuriteoutgrowth is then determined. By comparing the effect of eachconcentration of CHEC-9 on neurite outgrowth and neuron survival, anoptimum concentration for the specific neuron population is determined.

[0067] After the optimum in vitro concentration of CHEC-9 has beendetermined for a specific neuron population, an appropriate dosage maybe deduced by an in vivo assay on laboratory animals, such as rats. Anequivalent lesion in a primate or human would damage approximately15-fold more cortical tissue. The area of brain damage is determined bystandard imaging techniques, e.g., MRI. Therefore, that lesion cavitymust be treated with an approximately 15-fold greater amount of thefactor.

[0068] CHEC-9 may be administered in any effective dosage amountdetermined as set forth above. An exemplary dose is a subcutaneousadministration of 50-500 μg/kg of CHEC-9 peptide. This dose may beadministered immediately after, within 1 hour, 2 hours, 12 hours, or 1day of acute injury, or periodically in the case of a chronic condition.

[0069] A pharmaceutical preparation containing CHEC-9 may beadministered as a one-time dosage for cases of acute anoxia or trauma,or it may be administered at appropriate intervals in the case ofchronic degenerative disease, until the symptoms of the disease arereduced or eliminated. The appropriate interval of administration of thepharmaceutical preparation will depend on the type of neuron damagebeing treated and the condition of the patient.

[0070] The CHEC-9 peptide of the invention may be administered in linearor cyclized form. Additionally, the CHEC-9 peptide of the invention maybe administered in combination with another therapeutic agent, such as asteroid, a non-steroidal anti-inflammatory drug (NSAID), etc.

[0071] CHEC-9 Antibodies:

[0072] Purified CHEC-9 peptide, or fragments thereof, may be used toproduce polyclonal or monoclonal antibodies which also may serve assensitive detection reagents for the presence and accumulation of CHEC-9peptide (or complexes containing CHEC-9 peptide) in mammalian cells orbody fluids. Recombinant techniques enable expression of fusion proteinscontaining part or all of CHEC-9 peptide. The peptide may be used toadvantage to generate an array of monoclonal antibodies specific forCHEC-9, thereby providing even greater sensitivity for detection ofCHEC-9 in cells or body fluids.

[0073] Polyclonal or monoclonal antibodies immunologically specific forCHEC-9 peptide may be used in a variety of assays designed to detect andquantitate the peptide. Such assays include, but are not limited to: (1)flow cytometric analysis; (2) immunochemical detection/localization ofCHEC-9 peptide; and (3) immunoblot analysis (e.g., dot blot, Westernblot) of extracts from various cells. Additionally, as described above,anti-CHEC-9 antibodies can be used for purification of CHEC-9 peptideand any associated subunits (e.g., affinity column purification,immunoprecipitation).

[0074] Kits for Performing the Disclosed Methods:

[0075] In one broad aspect, the present invention encompasses kits foruse in administering CHEC-9. Such a kit may comprise a CHEC-9 peptide ina pharmaceutically acceptable excipient, such as artificial cerebralspinal fluid. The kit may also comprise devices which facilitateadministration of the peptide, such as catheters and syringes.

[0076] Further details regarding the practice of this invention are setforth in the following examples, which are provided for illustrativepurposes only and are in no way intended to limit the invention. Thefollowing materials and methods are provided to facilitate the practiceof the present invention.

EXAMPLE I Survival of Neural Cells is Supported by CHEC-9

[0077] A thirty amino acid N-terminal fragment of DSEP called Y-P30, wasoriginally purified from the culture medium of neural cell lines exposedto hydrogen peroxide. Y-P30 promotes neuron survival and inhibits theappearance and differentiation of monocyte derivatives(macrophages/microglia) in vitro and in vivo, including after systemicadministration (Cunningham, T J et al., 1998; Cunningham, T. J., et al.,2000). The cDNA and the gene location for full length human DSEP havebeen identified and encode a 12 kD secreted polypeptide. When thefull-length human protein is expressed in either mouse or human neuralcells, these cells become resistant to a variety of toxic treatments,including immune cell attack in xenocultures and in vivo (Cunningham TJ, et al., in press).

[0078] Based on the Y-P30 experiments, it was concluded that thesurvival and immune evasion activities of DSEP could be accomplished forthe most part by the N terminal 30 amino acids. However, the sequence ofthe secreted form of the native peptide differs from Y-P30 in that thelatter was made with cysteines at both positions 15 and 23 while thenative molecule contains only one cysteine at position 15 (with a lysineat position 23). In ongoing studies of biologically active forms ofY-P30, it was found that crosslinking the cysteines confers greatersurvival-promoting activity in vitro than a similar 30 amino acidfragment made without the K to C substitution, or a scrambled peptidewhere the amino acids (including those between the two cysteines) wereout of order. The K to C substitution therefore stabilizes an activeconformation of DSEP by allowing the formation of an intramoleculardisulphide bond. Therefore this part of the Y-P30 sequence —CHEASAAQC,designated herein as CHEC-9, was tested for DSEP/Y-P30-like activity.

[0079] Synthesis of Peptides.

[0080] Peptide synthesis was performed at the Protein ChemistryLaboratory in the Department of Pathology and Laboratory MedicineUniversity of Pennsylvania. The peptides were HPLC purified on a C18column, dried, reconstituted in water and dried again. Peptide stocksolutions (200-250 μg/ml, 218-273 μM) were prepared in 50 mM tris pH=7.4or DMEM and incubated at room temperature overnight or for 2 hrs at 37°.Free sulphydryls were measured using Ellman's reagent (DTNB, 0.04 mg/ml)in 0.1M NaH₂PO₄, 20 mM EDTA, pH=8 by mixing 25 μl sample with 275 μlreaction buffer. Absorbance of these samples was measured at 450 nm witha 808-xl microplate reader (Biotek Instruments), and was at backgroundlevels after cross-linking. In addition, the formation of intramoleculardisulphide bond in selected samples was verified by determining theexact molecular mass of the unfolded versus folded peptides usingelectrospray mass spectrometry (LC-ZQ Mass Spectrometer, Waters)

[0081] CHEC-9 Protects Neural Cells Subjected to Stress In Vitro

[0082] Various concentrations of CHEC-9 were tested in a stress testconsisting of medium change followed by serum deprivation (seeCunningham, et al., 2000, 2002). The CHEC-9 molecule was found to rescuecells when used at concentrations of 0.1 and 0.01 μM (10-100 picomolar).Optimal activity of the peptide was achieved after pre-incubation (250nM) with 10 mM adenosine triphosphate (Na-ATP) in a reaction mixturecontaining 120 mM KCl, 1 mM CaCl₂, 25 mM NaCl, and 25 mM tris (pH=7.4).This mixture was diluted in culture medium and then added to thestressed cells at the appropriate active picomolar concentrations.Vehicle treatment consisted of incubation buffer without the peptide atthe appropriate dilutions. Cell survival is measured by either countingsurviving attached cells or, as shown in FIG. 1, by colormetricdetermination after applying an electrocoupling reagent that responds tochemical reactions in normal cellular respiration. The number of neuronsprotected in the cultures is estimated to be between 3 and 10 fold,depending on the length of serum deprivation and the startingconcentration of cells.

[0083] CHEC-9 Protects Cerebral Cortex Neurons after Cortical StabWounds in Rats

[0084] Stab wounds were administered to the rostral cortical area 3 ofthe exposed cerebral cortex (with dura intact) of rats, using adissecting knife (blade=1×2 mm). The wound typically produces asignificant local inflammatory response, disruption of the functionallayers of the cortex, and marked atrophy and degeneration of neurons(FIG. 2, left panel). The principal immune cells involved in theinflammatory response are macrophages and microglia (FIG. 2, inset).Subcutaneous injection of CHEC-9 (0.4 mg/Kg bilaterally in the skin ofthe shoulder), 20 min after the placing of the wound, results in asignificant anatomical sparing of the perilesion parenchyma, as well asa more restricted inflammatory response (FIG. 2, right panel).

[0085] Activation of Microglia Cells is Inhibited by CHEC-9

[0086] Microglia cells were purified from neonatal rats according toestablished procedures and allowed to develop for an additional 72-96hrs in vitro, after which the cells are found to be 90-98% ED-1+. ED-1is a marker specific for rat microglia. Contaminating cells are GFAF+(suggesting they are astrocytes) or unreactive. TNFα immunoreactivity isat moderate to low levels in these cultures. If, however, the cells areactivated with 100 nM retinoic acid on days 1 and 2 in vitro andexamined on day 3 or 4, the ED-1 positive microglia cells displayrounded morphology with small or blunt processes suggesting that thecells are transformed into amoeboid microglia. (sometimes referred to asbrain macrophages, Milligan, et al 1991a;b). TNFα immunoreactivity ismore intense in these cultures. These same morphological changes havebeen described in several studies of microglial activation in vitro(e.g., Siao and Tsirka, 2002; Bothatschek, 2001). When the microglia aretreated with 1 nM CHEC-9 during the period of activation by RA (30 minafter the RA treatment), the cells in the CHEC-9 treated cultures are onaverage smaller with distinct processes suggesting the transformation tothe activated amoeboid morphology is inhibited (data not shown).Likewise, TNFα immunostaining in the cells is reduced. An experimentwith eight RA treated cultures is shown in FIG. 3. The 4 cultures in theright panels were also treated with 1 nM CHEC-9 peptide.

[0087] CHEC-9 Protects Neural Cells and Inhibits Inflammation

[0088] Animals, Surgery, and Histology.

[0089] All animal procedures were in compliance with the relevant lawsand institutional guidelines, and were approved by Animal Care and UseCommitteee of Drexel University College of Medicine. The lesion studieswere conducted on 15 long evans hooded rats weighing 225-275 g. Twelveof these rats were deeply anesthetized with ketamine/xylazine and placedin a sterotaxic holder. A 4×2 mm (rostrocaudal×mediolateral) skullopening was made on the right side starting just behind the coronalsuture and centered at a mediolateral position of +2.5 mm relative tobregma. A dissecting knife was penetrated through the dura and cortex inthe center of this skull opening to a depth of 1 mm. The skull defectwas filled with bone wax, the skin sutured closed, and the animal placedon a heating pad. Twenty minutes later, 0.4 cc of solution containing100 μg of peptide (˜0.4 mg/kg, 6 rats) or DMEM vehicle (6 rats) wasinjected under the skin of the shoulder near the midline. The rats wereperfused 4 days later and their brains processed for histology andimmunohistochemistry as in previous studies. Three rats were sacrificedwithout surgery or treatment. Alternate coronal sections of these brainswere stained with cresyl violet acetate and immunostained with the TUJ1antibody to neuronal specific tublin, isotype III (Covance ResearchProducts) or monocyte marker ED-1 Serotec. Secondary antibodies wereFITC or Rhodamine conjugated (Jackson Immunolabs). The density of ED-1+ameboid microglia in the perilesion parencyma wound was calculated afterexperimentally blinded counting of 4 fields (dorsal and ventral marginsof the wound) in 2 sections through the lesion.

[0090] Discussion

[0091] Four days following perfusion, there were no obvious behavioraldifferences between the treated and untreated groups. Both displayednormal locomotor activity and were alert and responsive to orientingstimuli. Cresyl violet-stained sections through the brains of controlrats revealed pronounced neuronal degeneration and accumulation of largenumbers inflammatory cells in the wound and in the parenchymasurrounding the wound. Immunostaining with the cell specific marker ED-1showed that many of these cells were macrophages and microglia. Themicroglia were activated and thus predominantly of the round ameboidtype. The cortical layers that are usually obvious in somatosensory area2 (where the laminae are distinctive) were no longer apparent because ofthe invasion of these normeuronal cells, and because of the frankdegeneration of the neurons. In rats injected with CHEC-9 both thedisruption of the cerebral cortex and accumulation of inflammatory cellsin the parechyma was inhibited. This effect was striking and apparent inall the rats treated with the peptide. The most obvious difference foundafter CHEC-9 treatment was the sparing of the cortical tissue adjacentto the wound in area 2. Granular and pyramidal neurons appeared of nearnormal size and distribution, and as a result, the cortical layers alsoappeared normal. In addition, rounded ED-1 positive cells weresignificantly reduced in the cortex. There were ED-1 reactive profilesscattered in the tissue near the lesion after CHEC-9 treatment, but thevast majority of these appeared to be processes of small, ramifiedcells, which is the morphology of nonactivated or “resting” microglia.

Example II CHEC-9 is a Potent Phospholipase A2 Inhibitor, and AlsoInhibits Paltelet Aggregation

[0092] Measurement of PLA2, Platelet Activity.

[0093] Trunk blood was collected from 16 additional Long Evans andSprague Dawley rats of both sexes following decapitation. Fourteen ofthese rats were paired according to strain, sex, and weight and injectedwith a control peptide/vehicle or with CHEC-9 forty-five minutes priorto sacrifice. Phospholipase A2 activity was determined in 10 rats andplatelets were isolated from the remaining 6 animals.

[0094] Serum samples and purified bee venom phospholipase were testedfor PLA2 activity using a 1,2-bis(heptanoylthio) glycerophosphocholinesubstrate (Caymen Chemical) which produces a DTNB reactive sulfhydrylupon cleavage of phospholipids at the at the sn-2 position (target ofall PLA2 enzymes). DTNB reactivity with serum, peptides, or PLA2 at theconcentrations used in these experiments was not detectable in theabsence of substrate (or vice versa). This substrate is sometimespreferred for inhibitor studies since with more natural substrates thereis the possibility for interfacial disruptions rather than trueinhibition (Mihelich E D, et al., 1997). All reactions were conducted intriplicate or quadruplicate in microwells at 25° with substrateconcentrations of 50-500 μM. The measurements were made on an ELX 808reader (Biotek Instruments) programmable for kinetic studies, andfurther analysis was performed using nonlinear regression software fromGraphpad which fit the data to a hyperbola (one site binding) fordetermining Vmax and Kd. For experiments with bee venom, CHEC-9, controlpeptides or tris solvent was mixed with equimolar sPLA2 and incubated at37° for 30 min. Platelets were isolated from whole blood treated with1.5 mM EDTA after gradient centrifugation in a 22:5 mixture of Trisglycine buffer and 60% iodixanol (OptiPrep, Axis Sheild). The plateletswere washed twice in Hanks balanced salt solution. Peptide was added inthe second wash if the animal was untreated, and after an additional 20min, the medium was collected and dialyzed overnight. Rates ofaggregation of the platelets were then determined in response toindicated concentrations of PMA in HBSS by the method of Bednar, et al,(1995) in which the absolute value of the rate of change of A₆₅₀ isproportional to rate of aggregation. The dialyzed supernatants weredried resuspended in SDS sample buffer and electrophoresced underreducing and non reducing conditions. Western blots were prepared as inprevious studies using a polyclonal antibody to sPLA2 IIa (CaymenChemical) that is reactive with rats platelet PLA2. Nonparametricstatistical analysis (Mann Whitney) was used throughout the study.

[0095] Discussion

[0096] Once its survival-promoting properties were recognized, CHEC-9was screened in several enzymatic and nonenzymatic assays related tocell survival and immunomodulation. In one of these, the peptide wasfound to inhibit activity of a secreted phospholipase A2 (sPLA2) derivedfrom bee venom. In these experiments, 70 nM of bee venom sPLA2 wasreacted with 50 μM of a glycerophosphocholine substrate in the presenceof various concentrations of CHEC-9 (FIG. 5A). The velocity of thereaction was measured and found to be reduced significantly by CHEC-9 atconcentrations of 100 nM and above. Next, the PLA2 enzyme activity ofserum from peptide and control-injected rats was compared in this sameassay after treating the rats according to the regimen used in thelesion studies. Rat serum shows significant phopholipase A2 activitythat appears to follow Michaelis-Menten kinetics, at least in the rangeof serum and substrate concentrations used in these experiments. Therewas inhibition of the serum PLA2 activity in rats injected with CHEC-9.FIG. 5B shows representative Michaelis-Menten plots usingpeptide-treated and control sera, including serum of rats treated with ascrambled version of the CHEC-9 peptide. In the latter experiments, itwas found that simply inverting the order of the glutamate and thealanine (that is E3-A4 to A3-E4) was sufficient to eliminate theinhibitory activity of CHEC-9. Analysis of kinetic plots fromCHEC-9-treated and control rats showed that the peptide, on average,reduced the maximum velocity of the reaction, however this effect wasvariable and not statistically significant (Vmax CHEC-9=70.3+7.4%, ofcontrols, p=0.132 n=6). The Kd of the reaction was increased in allpeptide treated rats, as much as 6-fold, and the difference betweenpeptide and control-treated rats was very significant (KdCHEC-9=313%+68% of controls, p=0.0087, n=6). These experiments provideevidence that the basis for the peptide's inhibition of PLA2 activity inserum is a reduction in the affinity of the enzyme(s) for substrateafter treatment.

[0097] Platelet activation is a PLA2-related activity and is alsoaffected by CHEC-9 treatment. When platelets are isolated from the bloodand then washed, they become activated and begin to aggregatespontaneously. It was observed that this spontaneous aggregation wasinhibited by prior treatment with CHEC-9, either using platelets frompeptide-injected rats or after direct treatment of isolated plateletswith 0.1 nM CHEC-9 (data not shown). If the platelets are then treatedwith phorbol-12-myristate-13-acetate (PMA) and agitated, plateletaggregation proceeds at a brisk rate for at least the next 5-30 min, andcan be monitored spectrophotometrically. This response is also inhibitedfor platelets treated with CHEC-9 directly or by injections into ratsprior to isolation (FIG. 5C). PMA is suggested to induce plateletactivation in concert with mobilization of intracellular calcium bystimulating phosphorylation of cytosolic PLA2 (McNicol A, et al., 1998).It is therefore possible that the peptide effects cytoplasmic PLA2directly, or indirectly through inhibition of secreted PLA2 enzymeswhich are released from activated platelets (Han W K, et al., 2003;Balboa M A, et al., 2003).

[0098] While there are likely to be several PLA2 isoforms present inserum, rat platelet sPLA2 (sPLA2 IIa) appears to be abundant in ratserum (Mihelich E D, et al., 1997; Hayakawa M, et al. 1987). The releaseof sPLA2 (IIa) from washed platelets was confirmed by Western blots ofplatelet supernatants. Interestingly, the sPLA (IIa) released byplatelets treated with CHEC-9 (either in the rat or in vitro) produced atypical bands on Western blots, migrating in SDS gels (under reducingconditions) with apparent molecular weights greater than the expected 14kD (FIG. 5D). The most prominent sPLA2 immunoreactive bands after CHEC-9treatment migrated above 40 kD, while control rats always showed a 14 kDband, and rarely showed higher molecular weight species. The finalposition of the sPLA2 bands after CHEC-9 treatment was variable fromsample to sample. However, the expected 14 kD species was not observedin CHEC-9 peptide-treated samples, suggesting that treatment hadmodified the enzyme structure and/or promoted the formation ofstabilized enzyme complexes or aggregates. Such aggregates might have alower affinity for substrate which would explain the kinetic differencesin PLA2 reactions found in peptide-treated rats.

[0099] Fatty acids, phospholipids, and other lipid mediators ofinflammation are increased following brain damage and inneurodegenerative diseases, and much of this increase results fromphospholipase A2 activity (Lipton P., 1999; Bazan N G, et al., 2002;Lukiw W J, et al., 2000). These products of lipid metabolism, along withthe coordinated activity of cytokines and other mediators, contributesignificantly to inflammation, and therefore also contributesignificantly to neuron death, either that which is observed after acutelesions to the CNS, or that found in many progressive neurodegenerativediseases. In addition, there are numerous examples of cross talk betweenlipid and cytokine mediated inflammatory responses that may amplifythese responses especially in the early stages of inflammation(Thommesen L, et al., 1998; Beck S, et al., 2003). Finally, thebreakdown of phospholipids by PLA2 enzymes produces major changes inmembrane function and signaling properties, and leads to increases infree fatty acid production and therefore free radical formation. Allthese changes are also potentially damaging to neurons and most othercell types.

[0100] These experiments indicate that CHEC-9 is effective to treat bothacute and chronic neurodegenerative and inflammatory conditions. Thepeptide's effects on other participants in PLA2-arachidonic acid pathwaymay prove interesting because many of these are the targets of potentialdrug therapies for inflammatory disorders in and outside the nervoussystem. On the other hand, one advantage of upstream inhibition of PLA2,presumably also an advantage for corticosteroids, is that upstreaminhibition eliminates the contribution of downstream functionallyredundant products or other participants in PLA2-directed metabolism,which may overcome the effects of drugs targeted downstream to morespecific elements in these pathways.

Example III Anti-Y-P30 Antibody Produces Increase in Cortical LesionSize and Sera Toxicity

[0101] This example demonstrates that there is a DSEP-like polypeptidein the rat brain that cross-reacts with affinity-purified polyclonalanti-human DSEP antibodies (FIG. 6). Rats were immunized with the Nterminal peptide of human DSEP (Y-P30) conjugated to Keyhole LimpetHemocyanin (KLH). Reactivity of their sera to DSEP was confirmed byWestern blots and ELISA. Small cortical lesions were produced in theimmunized rats. When the rats were sacrificed the extent of damage fromthe lesion, and the response of macrophages/microglia was tested.Additionally, rat antiserum was tested in a cell viability assay. It wasfound that DSEP immunized rats have exaggerated cortical lesions andincreased cytotoxicity of their sera.

[0102] Immunizations, Surgery, Adverse Reactions.

[0103] Injections and boosts were subcutaneous, bilaterally in theshoulder over a period of 1.5 months. Control rats were immunizedagainst KLH only. Serum titers of DSEP specific antibodies were measuredby ELISA. At the time of sacrifice, titers of the rats used in thisstudy were at least 1:1000 measured in multiple samples (data notshown). In addition, when DSEP antisera were tested in a cell killingassay they were found to be consistently more effective than KLHantisera (see below). Data was collected from 36 rats, 30 of which hadsmall lesions of cortical area 2 near the area 2/3 border. The lesion isproduced by stereotaxic positioning of a guide, opening the dura, andplacing a 1 mm³ piece of gelfoam on the cortical surface with lightpressure. Fifteen of these rats were immunized against DSEP-KLH and 15against KLH alone. Nine rats from each group were sacrificed 7 daysafter surgery and 6 were sacrificed after 4 days. Four immunized rats (2from each group) were sacrificed without surgery and two rats werenormal. There were no apparent adverse reactions to the immunizations ineither group. Gross behavior of all rats was similar. There was noevidence of increased inflammatory reactions peripherally or, aftersacrifice, in the CNS of immunized rats without surgery.

[0104] Lesion Volumes and ED1 Immunoreactivity

[0105] Four and 7 days following surgery, lesion volumes in anti-KLHrats were in a range that was consistent with parallel studies. Thedifferences in lesion sizes were not statistically significant in ratssurviving for 4 days (FIG. 6B). However at 7 days following surgery,lesion volumes in rats immunized against DSEP were more than 3-foldlarger (FIGS. 6A, 6B). The macrophage/microglial response to theselesions was examined after 7 days. As might be expected because of thelarger lesions, the anti-DSEP rats had an exaggerated appearance of ED1+cells at the margins of the lesion and in surviving deep white mattertracts surrounding the lesion (not shown).

[0106] Cell Viability Assay.

[0107] The antisera from rats immunized with DSEP-KLH were cytotoxic toboth HN33.1 and SY5Y cells. Anti-sera from 10 out of the 15 rats in eachgroup was tested at a 1:20 dilution. In all cases, the anti-DSEP serawere clearly more toxic to the cells than the control sera (FIG. 6C).During the first 24 hrs following treatment, sera from both groupscaused an apparent injury response and scattered degenerated cells,possibly due to complement-mediated mechanisms. Heating the sera for 30or 60 min at 55° to destroy complement had variable effects on thecultures but tended to improve this initial response. By 48-72 hrs thecells treated with anti-KLH sera had mostly recovered while the cellstreated with the anti-DSEP were degenerated completely.

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[0135] While certain of the preferred embodiments of the presentinvention have been described and specifically exemplified above, it isnot intended that the invention be limited to such embodiments. Variousmodifications may be made thereto without departing from the scope andspirit of the present invention, as set forth in the following claims.

What is claimed is:
 1. A neuron survival-promoting peptide having thesequence of CHEASAAQC (SEQ ID NO: 1) or a variant thereof.
 2. The neuronsurvival-promoting peptide of claim 1, said peptide having 1-9conservative amino acid substitutions.
 3. A pharmaceutical preparationcomprising the neuron survival-promoting peptide of claim 1, in abiologically acceptable carrier.
 4. A method for treating a patienthaving a neurodegenerative disorder, or disorder with an inflammatorycomponent comprising administering to said patient a therapeuticallyeffective amount of the peptide of claim
 1. 5. The method of claim 4,wherein said neurodegenerative disorder is selected from the groupconsisting of trauma, stroke, nonspecific anoxia, mental retardationsyndromes associated with progressive neuronal degeneration, and aneurodegenerative disease.
 6. The method of claim 5, wherein saidneurodegenerative disease is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, and amyotrophic lateralsclerosis (ALS).
 7. The method of claim 4, wherein said disorder with aninflammatory component is selected from the group consisting of asthma,autoimmune disease, and allergies.
 8. A method for treating a patienthaving an acute head trauma or neural injury comprising administering tosaid patient a therapeutically effective amount of the peptide ofclaim
 1. 9. The method of claim 8, wherein said peptide is administeredat a time point selected from the group consisting of within 1 hour ofinjury, within 2 hours of injury, within 6 hours of injury, within 12hours of injury, and within 1 day of injury.
 10. The method of claim 8,wherein said CHEC-9 peptide is administered in combination with anothertherapeutic agent.
 11. An isolated nucleic acid molecule encoding theCHEC-9 peptide of claim
 1. 12. An isolated RNA molecule transcribed fromthe nucleic acid of claim
 11. 13. An isolated plasmid comprising thenucleic acid molecule of claim
 11. 14. An isolated vector comprising thenucleic acid molecule of claim
 11. 15. An isolated retroviral vectorcomprising the nucleic acid molecule of claim
 11. 16. An isolated hostcell comprising the nucleic acid molecule of claim
 11. 17. The isolatedhost cell of claim 16, wherein said host cell is selected from the groupconsisting of bacterial, fungal, mammalian, insect and plant cells. 18.A host animal comprising the nucleic acid molecule of claim
 11. 19. Anantibody immunologically specific for the isolated CHEC-9 peptide ofclaim
 1. 20. The antibody of claim 19, which is a monoclonal antibody.21. The antibody of claim 19, which is a polyclonal antibody.
 22. A kitfor treating a neurodegenerative disorder or inflammatory disorder in apatient comprising: a) an isolated CHEC-9 peptide; b) a pharmaceuticalexcipient; and optionally c) a vehicle for administration, such as asyringe or catheter, and instructional material.
 23. The kit of claim22, wherein the kit further comprises a detectable label.
 24. A kit fordetecting CHEC-9 peptide comprising: a) means for isolating a CHEC-9peptide or nucleic acid encoding a CHEC-9 peptide from a biologicalsample; b) means for detecting and quantifying said peptides or nucleicacids; and optionally c) instructional material.